Next generation sequencing (NGS) has the potential to be an invaluable tool for the diagnosis and treatment of many different diseases and disorders. To reduce the cost of sequencing and the burden of data analysis, a target enrichment process can be used to increase the relative abundance of target sequences in a nucleic acid library prior to performance of the NGS reaction (e.g., as described US 2014/0287468, which is hereby incorporated by reference in its entirety).
Compatibility between target enrichment methods and multiplex sequencing processes is critical because the reduced complexity of enriched target libraries requires multiplex sequencing to be cost effective. Nearly all multiplex sequencing approaches include the labeling of individual libraries with a library-specific nucleic acid barcode, referred to as a “multiplex identifier nucleotide sequence,” or “MID,” through the addition of a MID to a platform-specific adapter sequence or a PCR primer. Because the sequence of the MID corresponds to the originating library, multiple libraries incorporating distinct MIDs can be combined and sequenced in a single sequencing reaction and, following sequencing, the MIDs can be used in silico to associate each resulting sequence with the library from which it originated.
In addition to MIDs, some target enrichment protocols also label individual DNA molecules with molecule-specific nucleic acid barcodes, referred to as “unique identifier sequences,” or “UlDs,” such as a degenerate base region (DBR), prior to amplification. The presence of such sequences makes it possible to distinguish unique DNA molecules from PCR duplicates, enabling the more accurate identification and quantification of unique DNA molecules and mutations.
As the diagnostic power of genome and transcriptome analysis increases, improved methods and compositions for the labeling of nucleic acid libraries with library-specific sequences and/or molecule-specific sequences are desirable to facilitate the use of target enrichment methods with multiplex NGS processes.